Thomas Marlovits

Structural and Systems Biology of Bacteria

"Integrating structure and systems at CSSB may allow us to discover, reveal and understand novel biological concepts."

Thomas Marlovits, CSSB Group Leader

Previous and current research

Molecular Machines in Action
A fundamental property of many biological processes is that they are performed by highly organized, multicomponent macromolecular assemblies, often referred to as molecular machines. The Marlovits group studies the structural basis for assembly, regulation, and function of transmembrane molecular machines. We use a multidisciplinary approach, by combining molecular biology, genetic, cellular, biochemical, and a wide-range of structural (EM, X-ray, NMR, X-linking/mass spectrometry) tools. We are developing novel imaging and modeling technologies to visualize dynamic molecular processes in unprecedented detail in situ and in action.

Microbial Pathogenesis
Gram-negative pathogens such as Yersinia, Shigella, Pseudomonas, enteropathogenic/enterohemorrhagic E. coli (EPEC/EHEC) and Salmonella are the causative agents for many diseases known to both animals and humans. These pathogens often originate as foodborne diseases and result in outcomes ranging from a mild stomach ache to death. Central to the pathogenicity are bacterial toxins ('effectors'), which are delivered via the type III secretion system, a large membrane embedded machinery, from the bacterium to its host cell. Once delivered, these translocated effector proteins have the remarkable capacity to modulate various host-cell pathways that induce membrane ruffling and subsequently make the host accessible to bacterial infection.

Type III secretion system: Unfolded protein transport across membranes
Our recent structural analysis (Schraidt & Marlovits, Science 2010) of the injectisome, the most prominent, cylindrical structure of the type III secretion system, revealed a potential secretion path through the central part of the membrane embedded complex. However, the inner diameter of this path is too small to accommodate a fully folded effector protein, suggesting that either the injectisome must undergo large conformational changes during transport or the effector proteins need to be unfolded.

To investigate the type III secretion of human pathogens, we focused (1) on determining the secretion path of injectisomes, (2) on understanding the mechanism of transport, and (3) on visualizing protein transport in situ. We discovered that substrates are inserted into the secretion path in a polar fashion - N-terminal regions first – and that they are transported in an unfolded state. To understand, whether such a behavior is in fact observed in situ, we analyzed protein transport across membranes in a near-native state using cryo electron tomography (Radics et al 2014). For the first time, we were able to visualize pathogenic type III secretion systems in action.
Technology Development - Atomic structure determination from lower resolution cryo-EM maps
Direct electron detectors are key to the recent revolution in structural biology and have made it possible to generate electron density maps at near atomic resolution using cryo electron microscopy from non-crystalline sample material. However, building accurate models into these 3-5Å maps remains a challenge. We recently developed a new modeling approach that integrates Monte Carlo optimization with local density guided moves, Rosetta all-atom refinement, and real space B-factor fitting, thus yielding accurate models from experimental maps for three different systems with resolutions as low as 4.5Å (DiMaio et al Nature Methods 2015). Based on increasing need within the scientific community, we expanded this work by developing easy-to -use modeling tools which build accurate models at the highest possible resolution from single particle electron microscopy maps.

Future Goals
By understanding the molecular mechanism of TTSS-mediated protein transport at the highest possible resolution, we hope to provide a basis for the development of novel therapeutic strategies that will either inhibit its activity or modify the system for targeted drug delivery.